German A. Roth

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.

Molecular mechanisms involved in interleukin 1-beta (IL-1β)-induced memory impairment. Modulation by alpha-melanocyte-stimulating hormone (α-MSH)

Pro-inflammatory cytokines can affect cognitive processes such as learning and memory. Particularly, interleukin-1β (IL-1β) influences the consolidation of hippocampus-dependent memories. We previously reported that administration of IL-1β in dorsal hippocampus impaired contextual fear memory consolidation. Different mechanisms have been implicated in the action of IL-1β on long-term potentiation (LTP), but the processes by which this inhibition occurs in vivo remain to be elucidated. We herein report that intrahippocampal injection of IL-1β induced a significant increase in p38 phosphorylation after contextual fear conditioning. Also, treatment with SB203580, an inhibitor of p38, reversed impairment induced by IL-1β on conditioned fear behavior, indicating that this MAPK would be involved in the effect of the cytokine. We also showed that IL-1β administration produced a decrease in glutamate release from dorsal hippocampus synaptosomes and that treatment with SB203580 partially reversed this effect. Our results indicated that IL-1β-induced impairment in memory consolidation could be mediated by a decrease in glutamate release. This hypothesis is sustained by the fact that treatment with d-cycloserine (DCS), a partial agonist of the NMDA receptor, reversed the effect of IL-1β on contextual fear memory. Furthermore, we demonstrated that IL-1β produced a temporal delay in ERK phosphorylation and that DCS administration reversed this effect. We also observed that intrahippocampal injection of IL-1β decreased BDNF expression after contextual fear conditioning. We previously demonstrated that α-MSH reversed the detrimental effect of IL-1β on memory consolidation. The present results demonstrate that α-MSH administration did not modify the decrease in glutamate release induced by IL-1β. However, intrahippocampal injection of α-MSH prevented the effect on ERK phosphorylation and BDNF expression induced by IL-1β after contextual fear conditioning. Therefore, in the present study we determine possible molecular mechanisms involved in the impairment induced by IL-1β on fear memory consolidation. We also established how this effect could be modulated by α-MSH.

Suppression of experimental autoimmune encephalomyelitis (EAE) by intraperitoneal administration of soluble myelin antigens in Wistar rats

Intraperitoneal (i.p.) treatment of Wistar rats with bovine myelin (BM) or myelin basic protein (MBP) previously to immunization with BM-CFA showed a diminished incidence and severity of experimental autoimmune encephalomyelitis (EAE) (2/13 and 0/7, respectively) when compared with rats immunized with BM-CFA (11/17) or i.p. treated with ovalbumin (2/4). Concomitantly, animals treated with BM or MBP exhibited a marked reduction of proliferative response to MBP which was highly positive when spleen mononuclear cells from nontreated and ovalbumin treated animals were assayed. Rats that were treated with MBP before immunization produce IgA, IgM, total IgG and subclasses of IgG, IgG2a, IgG2b, IgG2c specific for MBP in similar levels than those observed in nontreated immunized animals. However, a higher incidence and level of IgG1 was observed in MBP treated rats, meanwhile rats i.p. treated with total BM showed a highly reduced humoral response. The herein presented results show that i.p. treatment with low amounts of soluble forms of myelin antigens markedly reduced the clinical symptoms of the disease, the histological alterations, the cellular proliferative response to MBP, and produced changes in the autoimmune humoral response.

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.

Myelin basic protein domains involved in the interaction with actin

A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence although it is reached at the same MBP:actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.

Oral Testosterone in Male Rats and the Development of Experimental Autoimmune Encephalomyelitis

Objectives: Considering that sex steroids can influence the immune system, we studied the development of experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune disease of the central nervous system, and the concomitant cell-mediated immunity in gonadally intact and gonadectomized male Wistar rats given testosterone supplementation. Methods/Results: Sham-operated rats and surgically castrated animals were orally self-administered with vehicle or testosterone added in the water bottle for 20 days before EAE induction. The androgenic effect of oral testosterone self-administration was evidenced by changes in body weight, and in the weights of androgen-dependent testes and seminal vesicles. Testosterone administration reduced the incidence of clinical signs of EAE in sham-operated animals and reversed the clinical symptoms of the disease associated with castrated EAE animals. The clinical signs observed in the different groups correlated with changes in delayed-type hypersensitivity and mononuclear cell-proliferative responses to the encephalitogenic myelin basic protein. Moreover, testosterone but not cholesterol supplementation in vitro suppressed the proliferative response of mononuclear cells to myelin basic protein suggesting that testosterone may affect specific immune functions through direct actions on immune cells. Finally, self-administration of testosterone induced also elevated corticosterone levels that in sham-operated rats correlated with the low incidence of the disease and in gonadectomized animals could be involved in the remission of clinical symptoms of EAE. Conclusions: These results suggest that orally self-administered testosterone can modulate specific cellular immune responses and serum corticosterone levels leading to changes in the development of EAE.

Passive transfer of experimental autoimmune encephalomyelitis in Wistar rats: dissociation of clinical symptoms and biochemical alterations.

We have used passive transfer of myelin‐reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freunds adjuvant‐immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen‐specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE‐transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease. J. Neurosci. Res. 59:283–290, 2000

Inhibition of Ca2+-dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca2+‐dependent but not the Ca2+‐independent glutamate release induced by KCl and 4‐aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca2+ using fura‐2‐acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage‐dependent Ca2+ influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P‐site1. Our results show that the inhibition observed on the Ca2+‐dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca2+.

Experimental allergic encephalomyelitis: Dissociation of immunochemical and clinicopathological responses in two strains of rats

Abstract The differential expression of experimental allergic encephalomyelitis (EAE) was studied in two rat strains (Lewis and Wistar) sensitized with two different doses of myelin from guinea pig or bovine in complete Freunds adjuvant. The clinical development was more severe and the symptoms had an earlier onset in the Lewis than in the Wistar rats. Both strains of rats responded immunologically to the injected antigens. Quantitation of anti-MBP antibodies gave significant higher titers in the relatively resistant than in the susceptible rats. Conversely, widespread histopathological alterations were principally observed in the Lewis rats, whereas scarce infiltration and only in meninges was detected in Wistar rats. Autoimmunoglobulins fixed in infiltrated regions as well as in CNS cells were characteristic of Lewis rats with EAE but absent in all the Wistar rats. These cells were tentatively identified as motor neurons present in the spinal cord lateral motor columns and motor-controlling neurons in the lateral and spinal vestibular nucleus of the brainstem. The present results suggest that the different susceptibility to EAE in these two strains of rats could be due to a different immunological response that affect specific CNS cells.

Diphenyl diselenide-modulation of macrophage activation: Down-regulation of classical and alternative activation markers

Diphenyl diselenide (PhSe)2, a simple organoselenium compound, possesses interesting pharmacological properties that are under extensive research. As macrophages respond to microenvironmental stimuli and can display activities engaged in the initiation and the resolution of inflammation, in the present report we describe the ability of (PhSe)2 to modulate the macrophage activation. Our data indicate that (PhSe)2 could inhibit the NO production in a dose-dependent fashion in peritoneal macrophages activated by LPS or treated with vehicle alone. We could demonstrate that this effect correlated with a reduction in the expression of the inducible NO synthase in (PhSe)2-treated cells. Furthermore, (PhSe)2 suppressed the production of reactive oxygen species, diminished the activity of the arginase enzyme, and the accumulation of nitrotyrosine modified proteins in LPS-stimulated macrophages. This compound also diminished the antigen presentation capacity of classically activated macrophages, as it reduced MHCII and CD86 expression. In addition, (PhSe)2 modulated the alternative activation phenotype of macrophages. Dexamethasone-activated macrophages presented higher production of IL-10 and CD206, which were both down-regulated by the addition of (PhSe)2. These results suggest that (PhSe)2 possesses antioxidant and anti-inflammatory activities in classically-activated macrophages. We could demonstrate that (PhSe)2 can be also utilized to modulate the alternative activation phenotype of macrophages.